The concentration limit of detection cLOD varied from 0. Hence, blank injections between samples were not necessary. The analyte peak areas from the re-analyses were compared to the initial peak areas from day 1. The other neurotransmitters were stable at room temperature for at least 1 week. Several LC-MS methods 12 , 13 , 14 , 24 , 25 , 26 have been reported for polar NT determination, but disadvantages can be the need for extensive manual sample preparation and large sample amounts, etc.
For the more hydrophobic neuropeptides, standard reversed phase RP stationary phases have traditionally been used 27 , Disadvantages of such approaches are that they are limited to compounds of some hydrophobicity, and can therefore not be applied for small polar NT without the use of ion-pairing reagents typically not compatible with MS or chemical derivatization of the analytes significantly adding to time spent on sample preparation. To our knowledge, no previous reports have demonstrated the ability to simultaneously handle both very hydrophilic and hydrophobic NTs.
Hence, key advantages of the presented AFFL-SPE-cLC-MS platform include simultaneous handling of polar NTs as well as neuropeptides with enhanced sensitivity enabling analysis of small samples , a high degree of automation and increased speed. This was somewhat expected due to its rapid metabolism. On-line SPE also greatly reduces manual steps in sample preparation, which can be a central source of error in analysis To avoid this, the AFFL feature was employed, ensuring robust performance.
Whole blood samples are notorious for clogging LC equipment. We largely attribute the absence of problems analysing whole blood samples to the AFFL feature, which captures particulate matter with a filter that is back-flushed after each injection. This does not appear to be a significant issue when employing larger bore SPE-LC systems 37 , but becomes a factor with small-bore systems such as cLC , where system void volumes can play a larger role in system performance Based on this, ACN with 0.
Since whole blood could be analysed without plasmafication, additional time and manual work were saved, and the blood sample could be analysed less than one hour after the blood was collected. When performing targeted determination and quantifications, triple quadrupole 3Q instruments are often preferred, as they are known for their excellent quantitative capabilities. Orbitrap instruments are however superior 29 in resolution, and more typically used for untargeted analysis e.
This study exemplifies that precise quantifications of small metabolites can be successful with an Orbitrap instrument. In fact, linear calibration curves were obtained even without correction with internal standards Supplementary Fig. S4 , which implies relatively trustworthy quantifications for untargeted metabolomics as well. Considering that miniaturized HILIC-Orbitrap-based LC-MS systems work well for both proteomics 38 , 39 and NTs and likely other metabolites as described here, it is worth considering that a single instrument set-up similar to that described here can be used for both bottom-up proteomics and metabolomics, allowing their simultaneous determination.
Few methods on quantitative determination of NTs in human whole blood have been published, and reference materials are to the authors' knowledge, not available. However, the endogenous levels quantified by our method were comparable but somewhat higher to levels reported in plasma samples Supplementary Table S2. The reason is likely the high uptake of several neurotransmitters in blood platelets 40 , and indeed the levels of some of the analytes were lower in plasma, especially serotonin measured with a non-validated method.
The platform can be used for single-run chromatography of a wide range of NTs monoamines, amino acids and peptides. All methods were carried out in accordance with the approved guidelines and regulations. For validation studies, 8 blood samples were pooled prior to precipitation. For all chemicals and equipment used, see Supplementary Chemicals and equipment. S1 for an illustration of the entire system and Fig. The analytes will pass through the filter and be retained on the SPE column, while un-retained non-polar compounds and solvent will go directly to waste.
The purpose of the filter is to prevent larger particles from the sample e. In position 2 inject the 10 port valve is switched and the cLC pump elutes the analytes off the SPE column and onto the analytical column. The rest of the procedure was the same as for the sample preparation see section above. The calibration samples were made in the same way as the validation samples, and all samples were analysed on the same day as they were prepared. The within-day repeatabilities were found by analysing 6 sample replicates of validation samples with L, M and H concentration levels on the same day, while validation samples with L, M and H concentrations were analysed on 5 different days to investigate the between-day repeatabilities.
However, a crude estimate was calculated by extrapolation for details, see Supplementary note Calculations of cLOD. The recovery was investigated in the following way: one pooled blood sample was spiked with NT analytes XH level, to minimize contribution from endogenous levels before protein precipitation, and another pooled blood sample was spiked with NT analytes after protein precipitation. To calculate the recovery, equation 1 was used. A is the peak area of the analyte and A is is the peak area of the internal standard, respectively. To quantify the amounts of NTs in the whole blood samples, calibration curves based on calibration samples with concentrations from XL to XH Supplementary Table S4 , constructed using Excel, were used.
Since the calibration solutions were made by spiking blood which already contained the neurotransmitters of interest, the endogenous levels of NTs were calculated using the regression equation from the calibration curve, corrected for the endogenous level for details, see Supplementary note Calculation of NT concentrations, and Supplementary Fig.
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